US8785139B2ActiveUtilityA1

Modification-dependent activity assays

88
Assignee: WEBER ALFREDPriority: May 18, 2011Filed: May 18, 2012Granted: Jul 22, 2014
Est. expiryMay 18, 2031(~4.9 yrs left)· nominal 20-yr term from priority
Inventors:Alfred WeberAndrea EngelmaierHans-Peter Schwarz
A61P 37/02A61P 5/00A61P 43/00G01N 2440/00G01N 33/6842G01N 33/6857
88
PatentIndex Score
15
Cited by
7
References
28
Claims

Abstract

Disclosed herein are methods, systems and kits to measure the presence and/or activity of recombinant polypeptides comprising a modification.

Claims

exact text as granted — not AI-modified
The invention claimed is: 
     
       1. A method for detecting the presence of a recombinant polypeptide comprising a modification, the method comprising the steps of:
 incubating a sample including the recombinant polypeptide comprising the modification with a capture agent that selectively binds the modification under conditions allowing the selective binding of the capture agent to the modification, thereby forming a polypeptide-agent complex; 
 purifying the polypeptide-agent complex from the sample; and 
 assaying for the presence of the recombinant polypeptide and/or a polypeptide activity, wherein detection of the recombinant polypeptide and/or the polypeptide activity is indicative of the presence of the recombinant polypeptide comprising the modification. 
 
     
     
       2. The method according to  claim 1 , wherein the sample includes a polypeptide without the modification and/or a polypeptide with a different pattern of degree of modification. 
     
     
       3. The method according to  claim 1 , wherein the recombinant polypeptide is a therapeutic polypeptide. 
     
     
       4. The method according to  claim 1 , wherein the recombinant polypeptide is a coagulation factor. 
     
     
       5. The method according to  claim 1 , wherein the coagulation factor is a Factor II, a Factor IIa, a Factor VII, a Factor VIIa, a Factor VIII, a Factor VIIIa, a Factor IX, a Factor IXa, a Factor X, or a Factor Xa. 
     
     
       6. The method according to  claim 1 , wherein the capture agent has an association rate constant for a polypeptide comprising the modification of more than 1×10 5  M −1  s −1 . 
     
     
       7. The method according to  claim 1 , wherein the capture agent has a disassociation rate constant for a polypeptide comprising the modification of less than 1×10 −3  s −1 . 
     
     
       8. The method according to  claim 1 , wherein the capture agent has an equilibrium disassociation constant for a polypeptide comprising the modification of less than 0.500 nM. 
     
     
       9. The method according to  claim 1 , wherein the capture agent has an association rate constant for a polypeptide without a modification or a polypeptide with a different pattern or degree of modification of less than 1×10 4  M −1  s −1 . 
     
     
       10. The method according to  claim 1 , wherein the capture agent has an association rate constant (Ka) for the recombinant polypeptide comprising a modification that is more than 1×10 0  M −1  s −1  relative to the association rate constant (Ka) of the capture agent for a recombinant polypeptide without such a modification and/or the association rate constant (Ka) of the capture agent for a recombinant polypeptide with a different pattern or degree of modification. 
     
     
       11. The method according to  claim 1 , wherein the capture agent has an association rate constant (Ka) for the recombinant polypeptide comprising a modification that is at least 2-fold more then the association rate constant (Ka) of the capture agent for a recombinant polypeptide without such a modification and/or then the association rate constant (Ka) of the capture agent for a recombinant polypeptide with a different pattern or degree of modification. 
     
     
       12. The method according to  claim 1 , wherein the capture agent has a binding specificity ratio for a recombinant polypeptide comprising a modification relative to a recombinant polypeptide without such a modification and/or relative to a recombinant polypeptide with a different pattern or degree of modification of at least 2:1. 
     
     
       13. The method according to  claim 1 , wherein the capture agent distinguishes the recombinant polypeptide comprising a modification from the same polypeptide but without the modification. 
     
     
       14. The method according to  claim 1 , wherein the capture agent distinguishes the recombinant polypeptide comprising a modification from the same polypeptide but with a different pattern or degree of the same modification. 
     
     
       15. The method according to  claim 1 , wherein the recombinant polypeptide comprising the modification is a PEGylation Factor II, a PEGylation Factor IIa, a polysialylation Factor II, a polysialylation Factor IIa, a HESylation Factor II, a HESylation Factor IIa, a Sylation Factor II, or a Sylation Factor IIa. 
     
     
       16. The method according to  claim 1 , wherein the recombinant polypeptide comprising the modification is a PEGylation Factor VII, a PEGylation Factor VIIa, a polysialylation Factor VII, a polysialylation Factor VIIa, a HESylation Factor VII, a HESylation Factor VIIa, a Sylation Factor VII, or a Sylation Factor VIIa. 
     
     
       17. The method according to  claim 1 , wherein the recombinant polypeptide comprising the modification is a PEGylation Factor VIII, a PEGylation Factor Villa, a polysialylation Factor VIII, a polysialylation Factor Villa, a HESylation Factor VIII, a HESylation Factor Villa, a Sylation Factor VIII, or a Sylation Factor VIIIa. 
     
     
       18. The method according to  claim 1 , wherein the recombinant polypeptide comprising the modification is a PEGylation Factor IX, a PEGylation Factor IXa, a polysialylation Factor IX, a polysialylation Factor IXa, a HESylation Factor IX, a HESylation Factor IXa, a Sylation Factor IX, or a Sylation Factor IXa. 
     
     
       19. The method according to  claim 1 , wherein the capture agent is an anti-PEG antibody, an anti-PSA antibody, an anti-HES antibody, or an anti-S antibody. 
     
     
       20. The method according to  claim 1 , wherein the capture agent is attached to a solid support. 
     
     
       21. The method according to  claim 20 , wherein the solid support is a multi-well plate, a film, a tube, a sheet, a column, or a microparticle. 
     
     
       22. The method according to  claim 1 , wherein the assaying step is performed using a qualitative assay or a quantitative assay. 
     
     
       23. The method according to  claim 1 , wherein the assaying step is performed using an in vitro assay, a cell-based assay, or an in vivo assay. 
     
     
       24. The method according to  claim 1 , wherein the assaying step is performed using a non-specific polypeptide assay or a specific polypeptide assay. 
     
     
       25. The method according to  claim 24 , wherein the non-specific polypeptide assay is a UV absorption, a biuret assay, or a Bradford assay. 
     
     
       26. The method according to  claim 24 , wherein the specific polypeptide assay is a chromogenic assay, a colorimetirc assay, a chronometric assay, a chemiluminescense assay, an electrochemiluminescence assay, a bioluminescence assay, a fluorogenic assay, a resonance energy transfer assay, a plane polarization assay, a flow cytometry assay, an immuno-based assay or an activity assay. 
     
     
       27. The method according to  claim 24 , wherein the activity assay is an enzymatic activity assay, an inhibitory activity assay, a coagulation activity assay, or a polymerization activity assay. 
     
     
       28. The method according to  claim 1 , wherein selective binding of the capture agent occurs at a neutral to alkaline pH.

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